PROJECT SUMMARY C9FTD/ALS is a neurological disease caused by a mutation that results in expansion of a simple tandem repeat sequence in the gene C9ORF72. C9FTD/ALS is the leading genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease. Patients with this expansion exhibit the symptoms of FTD, ALS or both. These disorders are incurable and lack effective treatments. Although over two dozen distinct repeat expansion disorders exist, C9FTD/ALS bears mechanistic properties of many of them and is therefore a model for studying this class of disorders, as well as neurological disease in general. In C9FTD/ALS, the repeat expansion is transcribed into expanded tandem repeat-containing RNA, or xtrRNA, which is believed to mediate the molecular mechanisms of disease. C9ORF72 xtrRNA can aggregate in patient cell nuclei or be translated into repetitive poly-dipeptides in the cytoplasm. Both mechanisms have been proposed to contribute to disease. However, one outstanding question is why the mutant xtrRNA is not efficiently degraded in the nucleus to prevent aggregation or translation? Usually, unstable intronic or aberrant RNA in the nucleus is removed by nuclear RNA surveillance pathways. Surprisingly, almost nothing is known regarding the mechanisms used to clear xtrRNA from cells. In this project, C9FTD/ALS patient-derived cells are used to identify pathways and factors involved in nuclear RNA surveillance and turnover of C9ORF72 xtrRNA. The levels of candidate factors will be increased or decreased and the effect on xtrRNA aggregation, degradation, localization, and translation will be measured. Three specific aims are proposed. First, nucleases responsible for direct degradation of xtrRNA will be identified. It is hypothesized that the primary nuclease is the nuclear RNA exosome. The nuclear RNA exosome is known to require additional factors and targeting complexes to specify its substrates. Therefore, the second aim will investigate a set of candidate factors that could mediate degradation by the nuclear RNA exosome. Since exosome targeting complexes are often connected to cellular pathways and RNA types or species, the third aim will focus on the role of C9ORF72 intron removal and processing to understand how it dictates downstream degradation and turnover of xtrRNA. This project will identify and characterize the nuclear pathways and factors that recognize and target C9ORF72 xtrRNA for degradation. The premise of this project is simple but innovative. If successful, this project will open an entirely new area of investigation into repeat expansion disease mechanisms and make possible new strategies for developing therapeutic treatments for C9FTD/ALS and potentially other disorders.